miR-15b induced by platelet-derived growth factor signaling is required for vascular smooth muscle cell proliferation

The platelet-derived growth factor (PDGF) signaling pathway is essential for inducing a dedifferentiated state of vascular smooth muscle cells (VSMCs). Activation of PDGF inhibits smooth muscle cell (SMC)-specific gene expression and increases the rate of proliferation and migration, leading to dedifferentiation of VSMCs. Recently, microRNAs have been shown to play a critical role in the modulation of the VSMC phenotype in response to extracellular signals. However, little is known about microRNAs regulated by PDGF in VSMCs. Herein, we identify microRNA-15b (miR-15b) as a mediator of VSMC phenotype regulation upon PDGF signaling. We demonstrate that miR-15b is induced by PDGF in pulmonary artery smooth muscle cells and is critical for PDGF-mediated repression of SMC-specific genes. In addition, we show that miR-15b promotes cell proliferation. These results indicate that PDGF signaling regulates SMC-specific gene expression and cell proliferation by modulating the expression of miR-15b to induce a dedifferentiated state in the VSMCs. [BMB Reports 2013; 46(11): 550-554]     

Taurine suppresses platelet-derived growth factor (PDGF) BB-induced PDGF-beta receptor phosphorylation by protein tyrosine phosphatase-mediated dephosphorylation in vascular smooth muscle cells

In atherosclerosis, abnormal vascular smooth muscle cell (VSMC) proliferation plays an important role to form fibroproliferative lesions and platelet-derived growth factor (PDGF)-BB is one of the most potent chemoattractants and proliferative factors for VSMCs. Taurine, sulfur-containing beta-amino acid, has been considered to prevent the development of atherosclerosis, although the molecular mechanism remains obscure. Previously, we demonstrated that taurine significantly suppressed PDGF-BB-induced cell proliferation, DNA synthesis, immediate-early gene expressions and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in VSMCs. The present study was aimed at elucidating the precise molecular mechanism of taurine in PDGF-BB signaling pathway. We showed that taurine significantly suppressed PDGF-BB-induced phosphorylation of PDGF-beta receptor and activation of its downstream signaling molecules such as Ras, MAPK/ERK kinase (MEK)1/2 and Akt. Because taurine did not attenuate phorbol 12-myristate 13-acetate (PMA)-induced PDGF-beta receptor-independent ERK1/2 phosphorylation, we further investigated the suppressive mechanism of taurine in PDGF-beta receptor level. Although taurine did not directly affect PDGF receptor autophosphorylation in vitro, taurine promoted PDGF-beta receptor dephosphorylation and restored PDGF-BB-induced suppression of protein tyrosine phosphatase (PTPase) activity. Taken together, we propose that taurine could prevent or delay the progression of atherosclerosis by PTPase-mediated suppression of PDGF-beta receptor phosphorylation, and by decreasing the activation of its downstream signaling molecules in VSMCs.     

Akt regulates the survival of vascular smooth muscle cells via inhibition of FoxO3a and GSK3

Apoptosis of vascular smooth muscle cells (VSMCs) may lead to atherosclerotic plaque instability and rupture, resulting in myocardial infarction, stroke, and sudden death. However, the molecular mechanisms mediating survival of VSMCs in atherosclerotic plaques remain unknown. Although plaque VSMCs exhibit increased susceptibility to apoptosis and reduced expression of the IGF1 receptor (IGF1R) when compared with normal VSMCs, a causative effect has not been established. Here we show that increased expression of the IGF1R can rescue plaque VSMCs from oxidative stress-induced apoptosis, demonstrating that IGF-1 signaling is a critical regulator of VSMC survival. Akt mediates the majority of the IGF1R survival signaling, and ectopic activation of Akt was sufficient to protect VSMCs in vitro. Both IGF1R and phospho-Akt expression were reduced in human plaque (intimal) VSMCs when compared with medial VSMCs, suggesting that Akt mediates survival signaling in atherosclerosis. Importantly, downstream targets of Akt were identified that mediate its protective effect as inhibition of FoxO3a or GSK3 by Akt-dependent phosphorylation protected VSMCs in vitro. We conclude that Akt and its downstream targets FoxO3a and GSK3 regulate a survival pathway in VSMCs and that their deregulation due to a reduction of IGF1R signaling may promote apoptosis in atherosclerosis.     

Metabolic control analysis of the Warburg-effect in proliferating vascular smooth muscle cells

Summary The accumulation and proliferation of vascular smooth muscle cells (VSMC) within the vessel wall is an important pathogenic feature in the development of atherosclerosis. Glucose metabolism has been implicated to play an important role in this cellular mechanism. To further elucidate the role of glucose metabolism in atherogenesis, glycolysis and its regulation have been investigated in proliferating VSMC. Platelet derived growth factor (PDGF BB)-induced proliferation of VSMCs significantly stimulated glucose flux through glycolysis. Further evaluating the enzymatic regulation of this pathway, the analysis of flux:metabolite co-responses revealed that anaerobic glycolytic flux is controlled at different sites of gycolysis in proliferating VSMCs, being consistent with the concept of multisite modulation. These findings indicate that regulation of glycolytic flux in proliferating VSMCs differs from traditional concepts of metabolic control of the Embden–Meyerhof pathway.     

Effect of substrate stiffness and PDGF on the behavior of vascular smooth muscle cells: Implications for atherosclerosis

Vascular disease, such as atherosclerosis, is accompanied by changes in the mechanical properties of the vessel wall. Although altered mechanics is thought to contribute to disease progression, the molecular mechanisms whereby vessel wall stiffening could promote vascular occlusive disease remain unclear. It is well known that platelet‐derived growth factor (PDGF) is a major stimulus for the abnormal migration and proliferation of vascular smooth muscle cells (VSMCs) and contributes critically to vascular disease. Here we used engineered substrates with tunable mechanical properties to explore the effect of tissue stiffness on PDGF signaling in VSMCs as a potential mechanism whereby vessel wall stiffening could promote vascular disease. We found that substrate stiffness significantly enhanced PDGFR activity and VSMC proliferation. After ligand binding, PDGFR followed distinct routes of activation in cells cultured on stiff versus soft substrates, as demonstrated by differences in its intensity and duration of activation, sensitivity to cholesterol extracting agent, and plasma membrane localization. Our results suggest that stiffening of the vessel wall could actively promote pathogenesis of vascular disease by enhancing PDGFR signaling to drive VSMC growth and survival. J. Cell. Physiol. 225: 115–122, 2010. © 2010 Wiley‐Liss, Inc.     

microRNA let‐7g suppresses PDGF‐induced conversion of vascular smooth muscle cell into the synthetic phenotype

Platelet‐derived growth factor (PDGF) can promote vascular smooth muscle cells (VSMCs) to switch from the quiescent contractile phenotype to synthetic phenotype, which contributes to atherosclerosis. We aimed to investigate the role of microRNA let‐7g in phenotypic switching. Bioinformatics prediction was used to find let‐7g target genes in the PDGF/mitogen‐activated protein kinase kinase kinase 1 (MEKK1)/extracellular signal‐regulated kinase (ERK)/Krüppel‐like factor‐4 (KLF4) signalling pathway that affects VSMC phenotypic switching. The luciferase reporter assay and let‐7g transfection were used to confirm let‐7g target genes. Two contractile proteins alpha‐smooth muscle actin (α‐SMA) and calponin were VSMC‐specific genes and were measured as the indicators for VSMC phenotype. Lentivirus carrying the let‐7g gene was injected to apolipoprotein E knockout (apoE^?/?) mice to confirm let‐7g's effect on preventing atherosclerosis. Through the PDGF/MEKK1/ERK/KLF4 signalling pathway, PDGF‐BB can inhibit α‐SMA and calponin. The PDGFB and MEKK1 genes were predicted to harbour let‐7g binding sites, which were confirmed by our reporter assays. Transfection of let‐7g to VSMC also reduced PDGFB and MEKK1 levels. Moreover, we showed that let‐7g decreased phosphorylated‐ERK1/2 while had no effect on total ERK1/2. KLF4 can reduce VSMC‐specific gene expression by preventing myocardin–serum response factor (SRF) complex from associating with these gene promoters. The immunoprecipitation assay showed that let‐7g decreased the interaction between KLF4 and SRF. Further experiments demonstrated that let‐7g can increase α‐SMA and calponin levels to maintain VSMC in the contractile status. Injection of lentivirus carrying let‐7g gene increased let‐7g's levels in aorta and significantly decreased atherosclerotic plaques in the apoE^?/? mice. We demonstrated that let‐7g reduces the PDGF/MEKK1/ERK/KLF4 signalling to maintain VSMC in the contractile status, which further reduce VSMC atherosclerotic change.     

Mechanical Stretch Suppresses microRNA-145 Expression by Activating Extracellular Signal-Regulated Kinase 1/2 and Upregulating Angiotensin-Converting Enzyme to Alter Vascular Smooth Muscle Cell Phenotype

Phenotype modulation of vascular smooth muscle cells (VSMCs) plays an important role in the pathogenesis of various vascular diseases, including hypertension and atherosclerosis. Several microRNAs (miRNAs) were found involved in regulating the VSMC phenotype with platelet-derived growth factor (PDGF) treatment, but the role of miRNAs in the mechanical stretch-altered VSMC phenotype is not clear. Here, we identified miR-145 as a major miRNA contributing to stretch-altered VSMC phenotype by miRNA array, quantitative RT-PCR and gain- and loss-of-function methods. Our data demonstrated that 16% stretch suppressed miR-145 expression, with reduced expression of contractile markers of VSMCs cultured on collagenI; overexpression of miR-145 could partially recover the expression in stretched cells. Serum response factor (SRF), myocardin, and Kruppel-like factor 4 (KLF4) are major regulators of the VSMC phenotype. The effect of stretch on myocardin and KLF4 protein expression was altered by miR-145 mimics, but SRF expression was not affected. In addition, stretch-activated extracellular signal-regulated kinase 1/2 (ERK1/2) and up-regulated angiotensin-converting enzyme (ACE) were confirmed to be responsible for the inhibition of miR-145 expression. Mechanical stretch inhibits miR-145 expression by activating the ERK1/2 signaling pathway and promoting ACE expression, thus modulating the VSMC phenotype.     

MicroRNA-31 regulated by the extracellular regulated kinase is involved in vascular smooth muscle cell growth via large tumor suppressor homolog 2

Aberrant growth of vascular smooth muscle cells (VSMCs) is a major cellular event in the pathogenesis of many proliferative vascular diseases. Recently, microRNA-31 (miR-31) has been found to play a critical role in cancer cell proliferation. However, the biological role of miR-31 in VSMC growth and the mechanisms involved are currently unknown. In the present study, the expression of rat mature miR-31 (rno-miR-31) was determined in cultured VSMCs and in rat carotid arteries. We identified that rno-miR-31 is an abundant miRNA in VSMCs, and its expression was significantly increased in proliferative VSMCs and in vascular walls with neointimal growth. The up-regulation of rno-miR-31 in proliferative VSMCs was inhibited by the inhibitor of mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK). By both gain-of-function and loss-of-function approaches, we demonstrated that rno-miR-31 had a proproliferative effect on VSMCs. We further identified that LATS2 (large tumor suppressor homolog 2) is a downstream target gene product of rno-miR-31 that is involved in rno-miR-31-mediated effect on VSMC proliferation. The LATS2 as a target gene protein of rno-miR-31 is verified in vivo in balloon-injured rat carotid arteries. The results suggest that MAPK/ERK/miR-31/LATS2 may represent a novel signaling pathway in VSMC growth. miR-31 is able to enhance VSMC proliferation via its downstream target gene product, LATS2.     

FGFR1 forms an FRS2-dependent complex with mTOR to regulate smooth muscle marker gene expression

Vascular smooth muscle cells (VSMCs) switch from a contractile to a synthetic phenotype in human cardiovascular disease such as atherosclerosis and restenosis after angioplasty. VSMCs show reduced expression of contractile proteins and are capable of responding to mitogens by increasing expression of growth factor receptors. Fibroblast growth factor receptor-1 (FGFR1) signaling is one of several signaling pathways involved in this VSMC phenotypic switching. The aim of this study was to examine the signaling pathway downstream of FGFR1 in the regulation of SM marker gene expression. We found that FGFR1 activated Akt/mTOR pathway and that the mTOR inhibitor rapamycin partially reversed FGFR1-mediated downregulation of SM marker gene expression. Furthermore, we showed that mTOR forms a multi-protein complex with FGFR1 in VSMCs. These findings provide novel information for future development of therapeutic strategies for the treatment of human cardiovascular disease.     

TRPV1 activation impedes foam cell formation by inducing autophagy in oxLDL-treated vascular smooth muscle cells

Vascular smooth muscle cells (VSMCs) are an important origin of foam cells besides macrophages. The mechanisms underlying VSMC foam cell formation are relatively little known. Activation of transient receptor potential vanilloid subfamily 1 (TRPV1) and autophagy have a potential role in regulating foam cell formation. Our study demonstrated that autophagy protected against foam cell formation in oxidized low-density lipoprotein (oxLDL)-treated VSMCs; activation of TRPV1 by capsaicin rescued the autophagy impaired by oxLDL and activated autophagy–lysosome pathway in VSMCs; activation of TRPV1 by capsaicin impeded foam cell formation of VSMCs through autophagy induction; activation of TRPV1 by capsaicin induced autophagy through AMP-activated protein kinase (AMPK) signaling pathway. This study provides evidence that autophagy plays an important role in VSMC foam cell formation and highlights TRPV1 as a promising therapeutic target in atherosclerosis.     

ROCK1 induces ERK nuclear translocation in PDGF-BB-stimulated migration of rat vascular smooth muscle cells

It has been known that Rho-associated protein kinase (ROCK) signaling regulates the migration of vascular smooth muscle cells (VSMCs). However, the isoform-specific roles of ROCK and its underlying mechanism in VSMC migration are not well understood. The current study thus aimed to investigate the roles of ROCK1/2 and their relationship to the MAPK signaling pathway in platelet-derived growth factor (PDGF)-induced rat aorta VSMC migration by manipulating ROCK gene expression. The results revealed that ROCK1 small interfering ribonucleic acid (siRNA) rather than ROCK2 siRNA decreased PDGF-BB-generated VSMC migration, and upregulation of ROCK1 expression via transfection of constructed pEGFP-C1/ROCK1 plasmid further increased the migration of PDGF-BB-treated VSMCs. In PDGF-treated VSMCs, ROCK1 siRNA did not affect the phosphorylation levels of ERK and p38 in the cytoplasm, but decreased the level of ERK phosphorylation in the nucleus. These findings demonstrate that activated ROCK1 can promote VSMC migration through facilitating phosphorylation and nuclear translocation of ERK protein.     

CCN4 regulates vascular smooth muscle cell migration and proliferation

The migration and proliferation of vascular smooth muscle cells (VSMCs) are essential elements during the development of atherosclerosis and restenosis. An increasing number of studies have reported that extracellular matrix (ECM) proteins, including the CCN protein family, play a significant role in VSMC migration and proliferation. CCN4 is a member of the CCN protein family, which controls cell development and survival in multiple systems of the body. Here, we sought to determine whether CCN4 is involved in VSMC migration and proliferation. We examined the effect of CCN4 using rat cultured VSMCs. In cultured VSMCs, CCN4 stimulated the adhesion and migration of VSMCs in a dose-dependent manner, and this effect was blocked by an antibody for integrin α5β1. CCN4 expression was enhanced by the pro-inflammatory cytokine tumor necrosis factor α (TNF-α). Furthermore, knockdown of CCN4 by siRNA significantly inhibited the VSMC proliferation. CCN4 also could up-regulate the expression level of marker proteins of the VSMCs phenotype. Taken together, these results suggest that CCN4 is involved in the migration and proliferation of VSMCs. Inhibition of CCN4 may provide a promising strategy for the prevention of restenosis after vascular interventions.     

Manumycin A, inhibitor of ras farnesyltransferase, inhibits proliferation and migration of rat vascular smooth muscle cells.

Restenosis after angioplasty is thought to be caused by proliferation and migration of vascular smooth muscle cells (VSMCs), and it is a most serious problem in medical treatment. A low dose (50 ng/ml) of manumycin A, an inhibitor of p21(ras) (ras) farnesylation, significantly inhibited proliferation of rat VSMCs stimulated by the platelet-derived growth factor (PDGF). The mitoinhibitory effect of manumycin A was dose- and time-dependent but was independent of cell density. Western blot analysis showed that manumycin A reduced the amount of functional ras localized at the cytoplasmic membrane and inhibited the phosphorylation of p42/44 mitogen-activated protein kinase (MAPK). Manumycin A also inhibited VSMC migration and disorganized alpha actin fibers, as shown by immnofluorecence staining. These results indicate that the interruption of the ras/MAPK signal transduction pathway and the disorganization of alpha actin fibers are the main cause of manumycin A inhibition of VSMC proliferation and migration induced by PDGF.     

Aging differentially modulates the Wnt pro‐survival signalling pathways in vascular smooth muscle cells

We previously reported pro‐survival effects of Wnt3a and Wnt5a proteins in vascular smooth muscle cells (VSMCs). Wnt5a achieved this through induction of Wnt1‐inducible signalling pathway protein‐1 (WISP‐1) consequent to β‐catenin/CREB‐dependent, TCF‐independent, signalling. However, we found that as atherosclerosis advances, although Wnt5a protein was increased, WISP‐1 was reduced. We hypothesized this disconnect could be due to aging. In this study, we elucidate the mechanism underlying Wnt3a pro‐survival signalling and demonstrate the differential effect of age on Wnt3a‐ and Wnt5a‐mediated survival. We show Wnt3a protein was expressed in human atherosclerotic coronary arteries and co‐located with macrophages and VSMCs. Meanwhile, Wnt3a stimulation of primary mouse VSMCs increased β‐catenin nuclear translocation and TCF, but not CREB, activation. Wnt3a increased mRNA expression of the pro‐survival factor WISP‐2 in a TCF‐dependent manner. Functionally, β‐catenin/TCF inhibition or WISP‐2 neutralization significantly impaired Wnt3a‐mediated VSMC survival. WISP‐2 was upregulated in human atherosclerosis and partly co‐localized with Wnt3a. The pro‐survival action of Wnt3a was effective in VSMCs from young (2 month) and old (18–20 month) mice, whereas Wnt5a‐mediated rescue was impaired with age. Further investigation revealed that although Wnt5a induced β‐catenin nuclear translocation in VSMCs from both ages, CREB phosphorylation and WISP‐1 upregulation did not occur in old VSMCs. Unlike Wnt5a, pro‐survival Wnt3a signalling involves β‐catenin/TCF and WISP‐2. While Wnt3a‐mediated survival was unchanged with age, Wnt5a‐mediated survival was lost due to impaired CREB activation and WISP‐1 regulation. Greater understanding of the effect of age on Wnt signalling may identify targets to promote VSMC survival in elderly patients with atherosclerosis.     

Folic acid delays development of atherosclerosis in low‐density lipoprotein receptor‐deficient mice

Many studies support the cardioprotective effects of folic acid (FA). We aimed to evaluate the utility of FA supplementation in preventing the development of atherosclerotic in low‐density lipoprotein receptor‐deficient (LDLR?/?) mice and to elucidate the molecular processes underlying this effect. LDLR?/? mice were randomly distributed into four groups: control group, HF group, HF + FA group and the HF + RAPA group. vascular smooth muscle cells (VSMCs) were divided into the following four groups: control group, PDGF group, PDGF + FA group and PDGF + FA + RAPA group. Blood lipid levels, oxidative stress and inflammatory cytokines were measured. Atherosclerosis severity was evaluated with oil red O staining. Haematoxylin and eosin (H&E) staining was used to assess atherosclerosis progression. Immunohistochemical staining was performed with antismooth muscle α‐actin (α‐SMA) antibodies and anti‐osteopontin (OPN) antibodies that demonstrate VSMC dedifferentiation. The protein expression of α‐SMA, OPN and mechanistic target of rapamycin (mTOR)/p70S6K signalling was detected by Western blot analysis. FA and rapamycin reduced serum levels of total cholesterol, triacylglycerol, LDL, inhibiting oxidative stress and the inflammatory response. Oil red O and H&E staining demonstrated that FA and rapamycin inhibited atherosclerosis. FA and rapamycin treatment inhibited VSMC dedifferentiation in vitro and in vivo, and FA and rapamycin attenuated the mTOR/p70S6K signalling pathway. Our findings suggest that FA attenuates atherosclerosis development and inhibits VSMC dedifferentiation in high‐fat‐fed LDLR?/? mice by reduced lipid levels and inhibiting oxidative stress and the inflammatory response through mTOR/p70S6K signalling pathway.     

PYK2 signaling is required for PDGF-dependent vascular smooth muscle cell proliferation

Aberrant vascular smooth muscle cell (VSMC) growth is associated with many vascular diseases including atherosclerosis, hypertension, and restenosis. Platelet-derived growth factor-BB (PDGF) induces VSMC proliferation through control of cell cycle progression and protein and DNA synthesis. Multiple signaling cascades control VSMC growth, including members of the mitogen-activated protein kinase (MAPK) family as well as phosphatidylinositol 3-kinase (PI3K) and its downstream effector AKT/protein kinase B (PKB). Little is known about how these signals are integrated by mitogens and whether there are common receptor-proximal signaling control points that synchronize the execution of physiological growth functions. The nonreceptor proline-rich tyrosine kinase 2 (PYK2) is activated by a variety of growth factors and G protein receptor agonists in VSMC and lies upstream of both PI3K and MAPK cascades. The present study investigated the role of PYK2 in PDGF signaling in cultured rat aortic VSMC. PYK2 downregulation attenuated PDGF-dependent protein and DNA synthesis, which correlated with inhibition of AKT and extracellular signal-regulated kinases 1 and 2 (ERK1/2) but not p38 MAPK activation. Inhibition of PDGF-dependent protein kinase B (AKT) and ERK1/2 signaling by inhibitors of upstream kinases PI3K and MEK, respectively, as well as downregulation of PYK2 resulted in modulation of the G(1)/S phase of the cell cycle through inhibition of retinoblastoma protein (Rb) phosphorylation and cyclin D(1) expression, as well as p27(Kip) upregulation. Cell division kinase 2 (cdc2) phosphorylation at G(2)/M was also contingent on PDGF-dependent PI3K-AKT and ERK1/2 signaling. These data suggest that PYK2 is an important upstream mediator in PDGF-dependent signaling cascades that regulate VSMC proliferation.